This program explores innate immune, pro-inflammatory, and signaling functions of Nox family NADPH oxidases. The current research is focused on non-phagocytic oxidases (Nox1, Nox4, Duox1, Duox2) expressed primarily in epithelial cells, notably on mucosal surfaces (lung and gastrointestinal tract), in liver, kidney, thyroid and exocrine glands (salivary, mammary), and in vascular tissues. Deliberate reactive oxygen species (ROS) production by these enzymes can affect cell migration, proliferation, wound healing, tumor invasiveness and metastasis, cell differentiation, senescence, programmed cell death (apoptosis), oxygen sensing, extracellular matrix and thyroid hormone biosynthesis and responses to growth factors, cytokines, hormones and danger- and pathogen-associated molecular patterns (DAMPs and PAMPs). In some mucosal tissues (e.g., major airways) Duox enzymes produce sufficient hydrogen peroxide to support microbicidal peroxidases. In 2016, we explored functions of two epithelial NADPH oxidases in cell migration, proliferation and wound healing phenomena: 1) Nox1 regulation by peroxiredoxin 6 (Prdx6) and its role in supporting intestinal epithelial barrier functions and 2) Nox4 induction through the convergence of transformed growth factor-beta (TGF-beta) and mutant p53 signaling pathways and its role in tumor cell migration. Nox1, the closest homolog of the phagocyte (Nox2-based) oxidase, is most abundant in colon epithelial cells. Its activity is responsive to gut microbiota (i.e., lactobacillus species) through formyl peptide receptors, which can influence epithelial wound healing and homeostatic (barrier) functions. Like the phagocytic prototype, Nox1 depends of cytosolic regulators: Rac1, Nox organizer 1 (Noxo1, a p47phox homolog) and Nox activator 1 (Noxa1, a p67phox homolog). We identified Prdx6 as a novel binding partner of Noxa1, initially in yeast two-hybrid screening experiments using the C-terminal Noxa1 SH3 domain as bait. Prxd6 overexpression supports higher production of several Nox1 components, whereas Prdx6 silencing suppresses Nox1 protein levels and activity. We showed Prdx6 regulation of Nox1 occurs on at least two levels: 1) Prdx6-dependent stabilization of Nox proteins occurs through post-translational mechanisms, even in responses to TNF-alpha, and 2) Prdx6-dependent Nox1 activity appears to involve both its phosphoplipase A2 and peroxidase activities, since both lipase- and peroxidase-deficient mutants (Prdx6 C47S and S32A) fail to support higher Nox1 activity. Furthermore, we showed a Prdx6 phospholipase A2 transition-state substrate analogue (1-hexadecyl-3-(trifluoroethyl)-sn-glycero-2-phosphomethanol; MJ-33) inhibits Nox1 activity in three reconstituted cell models. Finally, we found that wound-healing responses of HCT-116 colon epithelial cells require concerted activities of Prdx6 and Nox1 for maximum cell migration and proliferation. These studies (published in Kwon, et al, Free Radic. Biol. Med. (2016)) highlight the potential of targeting Prdx6 to affect pro-inflammatory, proliferative and wound healing processes in intestinal epithelial cells that involve Nox1. Previous work has implicated Prdx6 phospholipase A2 and peroxidase activities in tumor growth and metastasis, while other independent studies indicate Nox1 promotes cancer cell proliferation. Our findings on the functional partnership of Nox1 with Prdx6 suggest novel approaches for therapeutic intervention in cancer progression. In other studies we found higher Nox4 expression correlates with the transformation of tumors to a metastatic phenotype. This year we expanded our observations supporting our hypothesis on the effects of TP53 mutations on Nox4 induction in several tumor models (lung, breast, hepatocyte, pancreatic). Here we examined effects of seven of the most commonly observed cancer-associated TP53 hotspot mutations (also detected in Li-Fraumeni syndrome patients), either by RNA interference of endogenous p53 mutant protein production or by heterologous expression of mutants in p53-null or wild type cell backgrounds. The mutant p53 forms all enhance Nox4 expression and metastatic cell migration in response to TGF-beta and Smad3, whereas wild type p53 suppresses these responses. We showed the p53 mutants promote higher Nox4 expression by two mechanisms: 1) they enhance Smad3 and p53 binding to regulatory elements identified in the Nox4 promoter and 2) they support higher histone-4 acetylation at these sites. Migration of metastatic lung and breast tumor cells by several p53 gain-of-function mutants was inhibited by dominant-negative forms of Nox4 and enhanced by histone deacetylase inhibitors. Together these findings suggest novel pharmacological, genetic and epigenetic approaches targeting Nox4 expression or activity may be used to inhibit metastasis of tumors with TP53 mutations. In related efforts aimed at examining the importance of Nox4 in metastatic and pro-fibrotic disease in whole animals, we have succeeded in creating Nox4-deficient mice using CRISPR/cas9 methodology. In other ongoing collaborative studies with NCBI investigators we are screening The Cancer Genome Atlas (TCGA) databases for independent evidence confirming correlations between TP53 mutation status and Nox4 induction in a variety of cancers.